1. Field of the Invention
The invention relates to membranes with immobilized microorganisms thereon and/or therein, to biofilm obtainable with such membranes, to a process for obtaining such membranes, and to reactors including said membranes and to a process involving the use of said membranes, in particular for the elimination of metals or of xenobiotic organic compounds.
2. Description of the Prior Art
Soluble metal removal is a technical challenge which must be met before the recycling process water or avoiding toxic discharges in plant effluent.
Heavy metals such as cadmium, lead, copper and zinc draw the attention of the hygienists because of their toxicity. The public health is directly concerned with the occurrence of heavy metals in water and soil even at low concentrations owing to their accumulation in vegetables through soil solution or contact with the contaminated soil, or with water that has leached the contaminated soil. In the same way, sewage sludges produced in biological waste treatment can be also loaded with heavy metals and with the presence of toxic and recalcitrant organic compounds (pesticides, PCB's, chlorinated aromatics, and the like). Sewage sludges are often used as fertilizers. Furthermore, there is a tendency towards water reuse and rivers as reservoirs for drinking water. It is the reason why national and international authorities have issued directives on limit value for heavy metals in industrial effluents as well as in aquatic systems.
So, it appears desirable that these health hazards should be avoided. For this purpose, the best way should be the removal of heavy metals at the emission point i.e. from the industrial effluents.
The removal of heavy metals from industrial wastewaters can be performed by several strategies. Among them, chemical precipitation by addition of hydroxides or calcium oxides, ion-exchange on resin or electrolyses are of common practices. These methods are used when rather large amounts of metals, i.e. more than 500 ppm, are involved. However, they are not appropriate to remove intermediate amounts of metals or to degrade xenobiotic organic compounds.
The use of ion exchange is more interesting at very low concentrations (less than about 5 ppm). But each of these methods has disadvantages. One disadvantage of ion exchange is high resin costs. One disadvantage of electrolyses is high energy costs. One disadvantage of hydroxides or calcium oxides is high sludge production.
It has also been investigated to use biomass which is immobilized in porous polysulfone beads for extracting toxic and heavy metals from dilute waste streams. The beads were fabricated from high-density polysulfone dissolved in dimethylformamide (DMF). Dried, thermally-killed biomass produced by algae, yeast, bacteria, and aquatic flora were blended into the polysulfone-DMF solution, and spherical beads were formed by injecting the mixture into water.
Contaminants removed from the waters using these beads included arsenic, cadmium, copper, mercury, lead, manganese, and zinc. Laboratory tests indicated that the beads may be especially useful in treating dilute wastewaters containing metal concentrations up to about 15 mg/l (cf. Jeffers T. H. et al., 1989, "Biosorption of metal contaminants using immobilized biomass", Biohydrometallurgy p. 317-327). This is a method applicable for very low concentrations of metal ions.
In this case, it is necessary to regenerate the bacteria, because there is adsorption of the metals to be removed, on the sites of the bacteria, and very little precipitation, and then the sites of the bacteria become saturated. When the bacteria have been regenerated, their efficiency to adsorb metal is lowered because all the sites cannot be regenerated or some sites are destroyed. No high upconcentration can be obtained.
Furthermore, it is necessary to use a large amount of beads, because it is possible to adsorb only between 5 and 10% of the metal with respect to the biomass, whereas if precipitation could take place with such beads (which is not the case), it would be possible to eliminate at least about 50% of metal with respect to the biomass.
Debus O. et al. ("Aerobic mineralization of benzene, toluene and xylenes by microorganisms attached to gas-permeable membranes", Technical University of Hamburg-Harburg, FRG, April 1990) have disclosed the use of pure or mixed cultures of microorganisms to biodegrade volatile organics like benzene, toluene, ethylbenzene and the isomeric xylenes (BTEX), under aerobic conditions. In order to avoid the conventional aeration systems producing a large number of bubbles, leading to a BTEX loss by stripping, gas permeable membranes are used, such as silicon rubber. The loss of volatile organics can be minimized by allowing BTEX-mineralizing microorganisms to colonize the membrane surface and form a barrier to the escaping substances.
In the Debus method, the bacteria receive only oxygen from the membranes. Moreover, the bacteria form a biofilm on the membranes, because the pores are too small for the bacteria to be immobilized on or in the membrane, and the nutrients are in the effluents, which involves a contamination of the effluents.
It is known that some microorganisms can immobilize metals up to high concentrations in their cellular materials specially when they are attached on a support. Among the culture devices which promote the growth of microorganisms on a surface, the biological fluidized bed is attractive because it has excellent adhesion potentialities for bacteria. The biological fluidized bed is composed of a cylinder packed with inert particles such as sand, anthracite, glass beads, plastic, stone gravels which provide support for microbial growth. The particles are freely suspended in the nutritive solution by an upward flow (cf. Remacle J. et al., Heidelberg 1983, "Uptake of heavy metals from industrial effluents by microorganisms developed in a biological fluidised bed" p. 936-939).
One of the drawbacks of existing biological fluidized beds is the fact that the nutritive medium for the microorganisms is mixed with the effluent to be treated, which involves a contamination of the effluent. Moreover, important amounts of carbon are needed, because they are added into the effluent. In addition, there is a big release of the microorganisms. Both these factors increase the cost of the treatment of the effluent.
Reticulated polyurethane foams are interesting supports allowing a high retention and an easy recovery of biomass just by squeezing (Cooper P. F. et al., 1986, in Process Engineering Aspect of Immobilised Cell Systems p. 205-217 Webb C. et al. (eds), I. Chem. Eng.). These are essential characteristics for the development of a process of metal recovery from industrial effluents.
In reticulated foams, the immobilized cell cultures were conducted in fixed bed microfermenters (V=0.41) continuously fed with a nutrient medium. The support particles consisted in 3.4 cm cubes with an internal porosity of 98% and a pore aperture of 30 ppi (Colombi Y. et al., 1987, "Cadmium uptake by Alcaligenes eutrophus immobilized in reticulated polyurethane foam", Proc. 4th European Congress on Biotechnology, 1: 120).
Reticulated foams have their disadvantages as well. For example, the biofilm is on the surface of the pores, which implies that there is a big release of cells. Besides, there is a need for important amounts of carbon.
During the last years, bacteria resistant to a variety of heavy metals were isolated and identified (Silver S. et al., 1988, "Plasmid-mediated heavy metal resistances" Ann. Rev. Microbiol. 42, 717-743). The mechanisms for such resistance are often controlled by plasmid borne genes or by transposons. A remarkable example of those resistant bacteria is Alcaligenes eutrophus var. metallotolerans. The representative strain CH34 was isolated in sediments from a decantation basin of a zinc factory (Mergeay M. et al., 1978, "Extrachromosomal inheritance controlling resistance to cadmium, cobalt and zinc ions: evidence from curing in a Pseudomonas" Arch. Int. Physiol. Biochim. 86, 440-441). Strain CH34 bears two large plasmids (Mergeay M. et al., 1985, "Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals". J. Bacteriol. 162 328-334) controlling resistance against Cd.sup.++, Co.sup.++, Zn.sup.++, Hg.sup.++, Tl.sup.+, Cu.sub.++, Pb.sup.++ (pMOL30, 240 kb) and Co.sup.++, Zn.sup.++, Ni.sup.++, Hg.sup.++, CrO4.sup.-, Tl.sup.+ (pMOL28, 165 kb) On pMOL28 nickel, cobalt and zinc genes (cnr) are [. . . ] on the same cluster very near to the chromate genes (chr) One DNA fragment (in pMOL30) of about 9 kb (Nies D. et al., 1987, "Cloning of plasmid genes encoding resistance to cadmium, zinc and cobalt in Alcaligenes eutrophus" Bacteriol. 16, 4865-4868) is responsible for the resistance against cadmium, zinc and cobalt (czc). And another gene cluster seems to code for copper and lead resistance (cup). Both plasmids contain a mercury transposon: Tn4378 and Tn4380 (Diels L. et al., 1985, "Mercury transposons from plasmids governing multiple resistance to heavy metals in Alcaligenes eutrophus CH34", Arch. Int. Physiol. Biochim. 93, B27-B28; Diels L. et al., 1989, "Large plasmids governing multiple resistances to heavy metals: a genetic approach" Toxic. Environm. Chem. 23, 79-89). Different heavy metal resistances genes are cloned and sequenced, namely czc (Nies D. H. et al., 1989, "Expression and nucleotide sequence of a plasmid determined divalent cation efflux system from Alcaligenes eutrophus", Proc. Natl. Acad. Sci. USA 86, 7351-7356), cnr (Siddiqui R. A. et al., 1989, "Cloning of pMOL28-encoded nickel resistance genes and expression of the gene in Alcaligenes eutrophus and Pseudomonas spp", J. Bacteriol. 171, 5071-5078) and chr (Nies A. et al., 1990, "Nucleotide sequence of chr genes responsible for resistance to chromate in Alcaligenes eutrophus", J. Biol. Chem. 265, 5648-5653). From the copper and lead genes, mutants are available.
The czc and mercury genes were used as probes for hybridization with total DNA from strains isolated from different mining and industrial sites in Belgium and Zaire (Diels L. et al., 1988, "Isolation and characterization of resistant bacteria to heavy metals from mining areas of Zaire", Arch. Int. Physiol. Biochim. 96, B13; Diels L. et al., 1988, "Detection of heterotrophic bacteria with plasmid-bound resistances to heavy metals from Belgian industrial sites", Arch. Int. Physiol. Biochim. 96, B14). From these different sites, strains hybridizing with these probes could be isolated (Diels L. et al., 1990, "DNA probe-mediated detection of resistant bacteria from soils highly polluted by heavy metals" Appl. Environm. Microbiol. 5, 1485-1491).
As could be shown by Nies D. et al. (1989, "Plasmid determined inducible efflux is responsible for resistance to cadmium, cobalt and zinc in Alcaligenes eutrophus" J. Bacteriol. 171, 896-900 and 1989, "Metal ion uptake by a plasmid-free metal sensitive Alcaligenes eutrophus strain", J. Bacteriol. 171, 4073-4075) resistance to chromate is inducible and based on decreased net accumulation of the metal anion. Resistance to zinc, cadmium, cobalt and nickel are resulting from inducible, energy dependent cation efflux systems. In some physiological circumstances Alcaligenes eutrophus can also accumulate and precipitate heavy metals (Diels L., 1990, "Accumulation and precipitation of Cd and Zn ions by Alcaligenes eutrophus strains", Biohydrometallurgy (1989) 369-377; Diels L. et al., 1989, "Isolation and identification of bacteria living in environments severely contaminated with heavy metals", 7th International Conference on Heavy Metals in the Environment, Sep. 12-15, 1989, Geneva). At increased concentrations of Cd or Zn ions, a removal of these metals from the solution is observed during the late log phase and the stationary phase. This accumulation and precipitation is correlated with the concentration and kind of carbon source (lactate or gluconate), with the progressive alkalinization of the periplasmic space and the surrounding medium, due to the proton antiporter system of the resistance mechanism, with the concentration of phosphate and appears to be associated with the outer cell membrane. The precipitation of CdCO.sub.3 and Cd(OH).sub.2 is proved by IR-spectroscopy. The interpretation of this feature is that the metal speciation will change at the cell surface due to the progressive pH increase, the steep pH gradient on this site, and the production of CO.sub.2 by the cell metabolism.
For electrochemical purposes, membranes have been developed (Leysen R. et al., 1987, "The use of heterogeneous membranes in electrochemical systems", in "Synthetic polymeric membranes", Eds. B. Sedlacek and J. Kahovec, W. de Gruyter, Berlin) composed of a polymeric polysulfone material in which zirconium oxide grains are distributed in a homogeneous way in order to form a composite membrane; these membranes are formed using the phase inversion technique (evaporation--crystallization).
This type of membrane has already been produced in three different configurations: flat membranes (Doyen W. et al., 1988, "The use of ZrO.sub.2 -based composite membranes for the separation of oil-water emulsions", in Proceedings of the Symposium on "Particle Technology in relation to Filtration Separation", Antwerp, October 3-4) with or without a reinforcing support, hollow fibers (Matthys J. et al., 1989, "Development of hollow fibers for the production of secretory products by living cells, in Proceedings of the Symposium on "Down stream processing in Biotechnology", Bruges, April 10-11) and tubes (Doyen W. et al., 1989, "New composite tubular membranes for ultrafiltration" in Proceedings of the "6th International Symposium on Synthetic Membranes in Science and Industry", Tubingen, September 4-8); however, the hollow fibers (when their diameter is smaller than about 4 mm) are very sensitive to clogging especially in the presence of an effluent which contains suspended materials.